New technologies accelerate the exploration of non-coding RNAs in horticultural plants.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs

MicroRNA-like RNAs from the same miRNA precursors play a role in cassava chilling responses

Abstract MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses

Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development

Emerging connections between small RNAs and phytohormones

Small RNAs (sRNAs), mainly including miRNAs and siRNAs, are ubiquitous in eukaryotes. sRNAs mostly negatively regulate gene expression via (post-)transcriptional gene silencing through DNA methylation, mRNA cleavage, or translation inhibition. The mechanisms of sRNA biogenesis and function in diverse biological processes, as well as the interactions between sRNAs and environmental factors, like (a)biotic stress, have been deeply explored. Phytohormones are central in the plant’s response to stress, and multiple recent studies highlight an emerging role for sRNAs in the direct response to, or the regulation of, plant hormonal pathways. In this review, we discuss recent progress on the unraveling of crossregulation between sRNAs and nine plant hormones

Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species

Transcription Factors and MicroRNA Interplay: A New Strategy for Crop Improvement

MicroRNAs (miRNAs) and transcription factors are master regulators of the cellular system. Plant genomes contain thousands of protein-coding and non-coding RNA genes; which are differentially expressed in different tissues at different times during growth and development. Complex regulatory networks that are controlled by transcription factors and microRNAs, which coordinate gene expression. Transcription factors, the key regulators of plant growth and development, are the targets of the miRNAs families. The combinatorial regulation of transcription factors and miRNAs guides the appropriate implementation of biological events and developmental processes. The resources on the regulatory cascades of transcription factors and miRNAs are available in the context of human diseases, but these resources are meager in case of plant diseases. On the other hand, it is also important to understand the cellular and physiological events needed to operate the miRNAs networks. The relationship between transcription factors and miRNA in different plant species described in this chapter will be of great interest to plant scientists, providing better insights into the mechanism of action and interactions among transcription factors (TFs) and miRNA networks culminating in improving key agronomic traits for crop improvement to meet the future global food demands

MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects

BACKGROUND A current challenge of microRNA (miRNA) research is the identification of biologically relevant miRNA:target gene relationships. In plants, high miRNA:target gene complementarity has enabled accurate target predictions, and slicing of target mRNAs has facilitated target validation through rapid amplification of 5' cDNA ends (5'-RACE) analysis. Together, these approaches have identified more than 20 targets potentially regulated by the deeply conserved miR159 family in Arabidopsis, including eight MYB genes with highly conserved miR159 target sites. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. Here, we examine the functional role of miR159 regulation for the other potential MYB target genes. RESULTS For these target genes, functional analysis failed to identify miR159 regulation that resulted in any major phenotypic impact, either at the morphological or molecular level. This appears to be mainly due to the quiescent nature of the remaining family member, MIR159c. Although its expression overlaps in a temporal and spatial cell-specific manner with a subset of these targets in anthers, the abundance of miR159c is extremely low and concomitantly a mir159c mutant displays no anther defects. Examination of potential miR159c targets with conserved miR159 binding sites found neither their spatial or temporal expression domains appeared miR159 regulated, despite the detection of miR159-guided cleavage products by 5'-RACE. Moreover, expression of a miR159-resistant target (mMYB101) resulted predominantly in plants that are indistinguishable from wild type. Plants that displayed altered morphological phenotypes were found to be ectopically expressing the mMYB101 transgene, and hence were misrepresentative of the in vivo functional role of miR159. CONCLUSIONS This study presents a novel explanation for a paradox common to plant and animal miRNA systems, where among many potential miRNA-target relationships usually only a few appear physiologically relevant. The identification of a quiescent miR159c:target gene regulatory module in anthers provides a likely rationale for the presence of conserved miR159 binding sites in many targets for which miR159 regulation has no obvious functional role. Remnants from the demise of such modules may lead to an overestimation of miRNA regulatory complexity when investigated using bioinformatic, 5'-RACE or transgenic approaches.RSA was funded by an ANU postgraduate scholarship and by a CSIRO Emerging Science Initiative. JL is the recipient of an ANU international student postgraduate scholarship. This research was supported by an Australian Research Council grant DP0773270

The miR159-GAMYB pathway: silencing and function of GAMYB homologues amongst diverse plant species

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression in eukaryotes. In plants, many miRNA families mediate silencing of target genes, which are involved in plant development and plant defence. For my thesis, I have been investigating the miR159-GAMYB pathway, which appears conserved from basal vascular plants to angiosperms. GAMYB transcription factors have been demonstrated to have conserved roles of programmed cell death (PCD) in both the seed aleurone and the anther tapetum in a number of different plant species. However, what the functional role of GAMYB is in vegetative tissues remains unknown. In Arabidopsis, miR159 is critical for proper growth, as its inhibition has a strong negative impact on vegetative growth, due to deregulated GAMYB expression. However, gamyb loss-of-functional mutants display a wild-type phenotype, as their expression is silenced to phenotypically inconsequential levels by miR159 in vegetative tissues. This raises two questions: (1) how is GAMYB so strongly silenced; (2) why is GAMYB strongly and widely transcribed in vegetative tissues for it to be then completely repressed by miR159? These two questions were the focus of my thesis. Firstly, how the Arabidopsis MYB33 and MYB65 are so strongly silenced in the model plant Arabidopsis was investigated. Both genes were predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site, composed of two stem-loop (SL) structures; whereas such SL structures were not predicted to form in other GAMYB-like genes that are targeted less efficiently by miR159 for expression regulation. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing mutations to form an artificial stem-loop structure adjacent to a miRNA-binding site restored strong miR159-mediated silencing. Although how these predicted structures promote miR159-mediated silencing are not determined, we speculate that the stem-loop structures in the vicinity of the miR159 binding site promotes accessibility of the binding site, where if adjacent sequences form strong stem structures, they are less likely to base-pair with binding site nucleotides, maintaining high accessibility of the binding site. Interestingly, the RNA SL structures are predicted to reside in GAMYB-like homologues of numerous angiosperm and gymnosperm plant species, arguing that these structures have been integral in the miR159-GAMYB regulatory relationship over a long period of time. In addition, these structures are not present in the Arabidopsis GAMYB-like homologues that are not transcribed in vegetative tissues, suggesting that selection for such structures only occurs for homologues transcribed in vegetative tissues as to prevent their expression and demarcating them as sensitive targets of miR159. Secondly, to investigate the functional role of the miR159-GAMYB pathway, target MIMIC159 (MIM159) transgenes, which can inhibit endogenous miR159 activity, were expressed in a number of Arabidopsis ecotypes, as well as in tobacco and rice. Inhibiting miR159 in all three plant species resulted in similar phenotypic outcomes, predominantly stunted growth and irregular leaf shape. This implies that the function and expression of the miR159-GAMYB pathway is strongly conserved in distant plant species. This raises several questions: why is GAMYB widely transcribed if its expression is strongly silenced by miR159 throughout the plant to result in little to no phenotypic impact; and why has this been strongly conserved across multiple plant species. When miR159 activity is inhibited in MIM159 tobacco leaves, pathways related to plant defence response are most up-regulated. This included PATHOGENESIS-RELATED PROTEIN (PR) mRNA levels that were 100-1000s fold up-regulated compared to wild type, and correlated with deregulated GAMYB expression. This finding suggests that the miR159-GAMYB pathway is involved in the plant defence response to biotic stress. However, PR expression is not up-regulated in Arabidopsis or rice when miR159 is inhibited, suggesting that despite the conserved nature of the miR159-GAMYB pathway, there are species-specific differences in its function

Genome-Wide Analysis of microRNA Expression Profile in Roots and Leaves of Three Wheat Cultivars under Water and Drought Conditions

The following are available online at https://www.mdpi.com/article/ 10.3390/biom13030440/s1. Figure S1: Fraction of different RNA species. Figure S2: Read length distribution of all genome mapped reads (a) from total reads (redundant reads) and (b) from unique reads (non-redundant reads. Figure S3: Library normalized RPM values distribution per sample of novel miRNAs. Figure S4: qRT-PCR analysis of the expression of novel miRNA Tae-mir-novel54-5p and known miRNA Tae-miR827c in 10 samples. Figure S5: Network analysis of (a) target genes by drought downregulated miRNAs and (b) drought upregulated miRNAs in leaves. Table S1: Quality and read mapping report. Table S2: Fraction of different RNA species. Table S3: Read length distribution of all genome mapped reads from total reads (redundant reads). Table S4: Read length distribution of all genome mapped reads from unique reads (non-redundant reads. Table S5: All miRNAs expression matrix. Table S6: Expression matrix of all the miRNAs in the SRA datasets. Table S7: miRNA expression matrix of all miRNAs in the Zea mays SRA datasets. Table S8: Degradome based target-gene predicted interactions. Table S9: qRT-PCR assay information. Table S10: Enrichment of functional annotations in miRNA target genes. Table S11: Mature and hairpin sequences of predicted miRNAs. Table S12: Degradome miRNA-target interaction predictions using CleaveLand4.Wheat is one of the most important food sources on Earth. MicroRNAs (miRNAs) play important roles in wheat productivity. To identify wheat miRNAs as well as their expression profiles under drought condition, we constructed and sequenced small RNA (sRNA) libraries from the leaves and roots of three wheat cultivars (Kukri, RAC875 and Excalibur) under water and drought conditions. A total of 636 known miRNAs and 294 novel miRNAs were identified, of which 34 miRNAs were tissue- or cultivar-specific. Among these, 314 were significantly regulated under drought conditions. miRNAs that were drought-regulated in all cultivars displayed notably higher expression than those that responded in a cultivar-specific manner. Cultivar-specific drought response miRNAs were mainly detected in roots and showed significantly different drought regulations between cultivars. By using wheat degradome library, 6619 target genes were identified. Many target genes were strongly enriched for protein domains, such as MEKHLA, that play roles in drought response. Targeting analysis showed that drought-downregulated miRNAs targeted more genes than drought- upregulated miRNAs. Furthermore, such genes had more important functions. Additionally, the genes targeted by drought-downregulated miRNAs had multiple interactions with each other, while the genes targeted by drought-upregulated miRNAs had no interactions. Our data provide valuable information on wheat miRNA expression profiles and potential functions in different tissues, cultivars and drought conditions

A cotton miRNA is involved in regulation of plant response to salt stress

The present study functionally identified a new microRNA (microRNA ovual line 5, miRNVL5) with its target gene GhCHR from cotton (Gossypium hirsutum). The sequence of miRNVL5 precursor is 104 nt long, with a well developed secondary structure. GhCHR contains two DC1 and three PHD Cys/His-rich domains, suggesting that GhCHR encodes a zinc-finger domain-containing transcription factor. miRNVL5 and GhCHR express at various developmental stages of cotton. Under salt stress (50–400 mM NaCl), miRNVL5 expression was repressed, with concomitant high expression of GhCHR in cotton seedlings. Ectopic expression of GhCHR in Arabidopsis conferred salt stress tolerance by reducing Na+ accumulation in plants and improving primary root growth and biomass. Interestingly, Arabidopsis constitutively expressing miRNVL5 showed hypersensitivity to salt stress. A GhCHR orthorlous gene At2g44380 from Arabidopsis that can be cleaved by miRNVL5 was identified by degradome sequencing, but no confidential miRNVL5 homologs in Arabidopsis have been identified. Microarray analysis of miRNVL5 transgenic Arabidopsis showed six downstream genes (CBF1, CBF2, CBF3, ERF4, AT3G22920, and AT3G49200), which were induced by salt stress in wild-type but repressed in miRNVL5-expressing Arabidopsis. These results indicate that miRNVL5 is involved in regulation of plant response to salt stress